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Postantibiotic Effect of Amikacin and Rifapentine against Mycobacterium avium Complex Luiz E. Bermudez, Martin Wu, Lowell S. Young, and Clark B. Inderlied
Kuzell Institute for Arthritis and Infectious Diseases. Medical Research Institute ofSan Francisco at California Pacific Medical Center; Department ofPathology and Laboratory Medicine. Children's Hospital of Los Angeles. University ofSouthern California
Early clinical experience with penicillin suggested that many infections could be treated adequately with intermittent dosing, even though drug levels in blood and tissue fell below inhibitory concentrations for several hours. Daily administration of only one or two large doses produces high peak and low (sometimes undetectable) trough concentrations of the drug. Studies within the last decade have provided evidence that in many situations pulse dosing works as well as or better than continuous administration of antibiotic, suggesting a postantibiotic effect (PAE) of the drug. The success of intermittent therapy with penicillin was attributed to the persistent effect of the antibiotic on surviving organisms. This persistent suppression of bacterial growth was then attributed to a lag in recovery of growth, a PAE that could take hours. Studies with a number ofgram-positive and gram-negative organisms have demonstrated the PAE of several antibiotics [I, 2). More recently, the efficacy of several antibiotics for treating bacterial infections in immunocompromised patients has been correlated with PAEs [3]. To date, PAE has been measured only against organisms that cause extracellular infections, but it is possible that the ability ofan antibiotic to inhibit growth ofintracellular bacteria is also related to the PAE. For instance, amikacin is active in vitro and in vivo against Mycobacterium avium even though serum levels exceed the MIC for only a few hours [4].
Received 29 January 1992; revised 4 May ·1992. Grant support: National Institutes of Health (AI-72637). Reprints or correspondence: Dr. Luiz E. Bermudez. Kuzell Institute. 2200 Webster s.. Rm. 305. San Francisco. CA 94115. The Journal of Infectious Diseases 1992;166:923-6 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0038$01.00
In this study we compared the PAE ofamikacin and rifapentine (known to concentrate intracellularly) on growth of M. avium.
Materials and Methods Bacteria. M. avium complex strain 101 (serotype 1) was isolated from a patient with AIDS. Strain 101 is virulent, as demonstrated by LD so studies in animals. Bacteria were cultured in Middlebrook agar 7H 10 medium (Difco, Detroit) for 10 days at 37°C. Morphologically similar colonies were harvested and resuspended in 7H9 broth supplemented with OADC (oleic acid, albumin, dextrose, and catalase; Difco). Bacteria were allowed to grow for 5 days. Tubes were maintained in stationary conditions but were slightly agitated by inversion twice a day. The day ofthe experiment, log-phase organisms were washed by centrifugation and resuspended in 7H9 broth containing antibiotics (I X, 4X, 8X MIC). Antibiotics. Amikacin (provided by Bristol Laboratories, Syracuse. NY) was diluted in phosphate buffer, pH 7.2, and stored in portions at a concentration of 1000 Jlg/mL at - 70°C. Rifapentine was provided by Le Petit, Milan, Italy. After dilution, 500 Jlg/mL suspensions were stored at -70°C up to I week. MICs, determined according to previously described protocol [5], were 4 Jlg/mL for amikacin and 2 Jlg/mL for rifapentine. Exposure to antibiotics and removal. At time zero an M. avium suspension containing either 104 or 107 cfu/rnl. was exposed to the antibiotic (10 mL of the bacterial suspension was treated with the drug to obtain the desirable final concentration of the antibiotic). After incubation for 15,30,60, and 120 min at 37°C, the tubes were centrifuged (2000 g. 15 min) and the bacterial pellet was resuspended in PBS. Bacteria were washed three times to ensure complete removal of the antibiotics and, after the third wash, resuspended in 7H9 broth with OADC. Removal of90% of supernatant in two washings reduces antimicrobial concentrations 100-fold [6].
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Postantibiotic effect (PAE) has received little attention in the therapy of chronic intracellular infections, such as those caused by mycobacteria. Amikacin is active therapeutically against Mycobacterium avium complex, even though serum levels exceed the MIC for only a few hours. To determine the PAE of amikacin and rifapentine for M. avium, bacteria were exposed to concentrations of IX, 4X, and lOX the MIC of each drug for up to 120 min. Regrowth of M. avium was compared with similarly diluted untreated cultures. No PAE was observed on an inoculum of 104 bacteria when rifapentine was used at 5X MIC, although a slight inhibition of growth was obtained at tux MIC for 2 h. For amikacin, PAE was observed up to 48 h at concentrations of 4X and sx MIC and exposure times of 30-120 min. A PAE of 22 h was seen with 107 cfu of M. avium during incubation for 30 min with amikacin at 4x MIC. These results show that amikacin, unlike rifapentine, has a long PAE against M. avium.
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Counts of viable bacteria. Counts of viable bacteria were made after plating onto 7H 10 agar plates as previously reported [7]. To obtain uniform bacterial suspensions, resuspended bacterial suspensions were vortex agitated for 1 min before plating. The number of viable organisms was determined at different time points between zero and 96 h after removal of the antibiotic. In some experiments, quantitative plate counts were done before and after centrifugation to rule out an effect of three washes on bacterial counts. No significant difference was observed between the number of viable bacteria before and after centrifugation (data not shown). Definition. PAE was defined as the difference in time required for a culture to increase by I log after antibiotic exposure minus that time for unexposed bacteria.
Effect of exposure to MIC of amikacin and rifapentine on growth of M. avium. To determine if exposure of M. avium strain 101 to MIC of amikacin or rifapentine had any persistent effect on bacterial growth, a suspension of 104 cfu of M. avium was incubated with MICs of amikacin or rifapentine (4 and 2 ~g/mL, respectively) for 30, 60, and 120 min. Antibiotics were then removed by washing, and the bacteria were cultured on medium without antibiotic. As shown in table I, exposure to amikacin for 30 or 60 min was associated with PAE of6 h (36 h to reach I log growth compared with 30 h for bacteria not treated with drugs). Exposure for 120 min was associated with PAE of 10 h. In contrast, exposure of M. avium to rifapentine for 30, 60, or 120 min was not associated with persistent inhibition of growth. Effect ofantibiotic concentration on PAE. Table I shows that the increase in concentration of antibiotics prolonged the persistent effect ofamikacin and rifapentine on M. avium growth. Incubation of M. avium with amikacin for 30 min at a concentration 4X the MIC had a PAE of 18 h, while incubating M. avium with rifapentine (5X the MIC) for 30 min was associated with a PAE of only 4 h. Of note, when the amikacin concentration was increased to 4X or 8X the MIC, there was no significant increase in the PAE. For example, the PAE for bacteria exposed to 4X the MIC ofamikacin for 30 min was 18 h compared with 20 h when the bacteria were exposed to a concentration of 8X the MIC ofamikacin. Similar results were observed when M. avium was exposed to 4X or 8X the MIC ofamikacin for 60 min. A significant increase in PAE was observed only when M. avium was exposed to sx the MIC for 120 min (PAE, 48 h) in comparison to 4X the MIC (PAE, 28 h). Similarly, exposure of M. avium to increased concentrations of rifapentine (lOX MIC) was not associated with increased persistence of antibiotic effect when compared with exposure to rifapentine at Sx the MIC. Effect ofnumbers ofbacteria in inoculum on PA£. To determine whether increased numbers of bacteria in the inoculum would have any effect on the persistence of the amikacin
Table 1. Postantibioticeffect (PAE) ofamikacin and rifapentine on M. avium strain 101 (104 organisms) with various concentra-
tions and durations of exposure. Duration of exposure (min). antibiotic (concentration) 30. Control M. avium (no antibiotic) Amikacin (MIC) Amikacin (4X MIC) Amikacin (8X MIC) Rifapentine (MIC) Rifapentine (5X MIC) Rifapentine ( lOX MIC) 60. Amikacin (MIC) Amikacin (4X MIC) Amikacin (8X MIC) Rifapentine (MIC) Rifapentine (5X MIC) Rifapentine ( IOx MIC) 120. Amikacin (MIC) Amikacin (4X MIC) Amikacin (8X MIC) Rifapentine (MIC) Rifapentine (5X MIC) Rifapentine ( IOx MIC)
Time (h) to grow Ilog* 30 36 48 50 30 30 30 36
PAE (h)
o 6 18 20
No. of experiments
5 3 3 3
o o o
2
6
3
53
23
60
30
30 34 34 40 58 78 30 34 34
o
3 3 2 2 2
4 4
10 28 48
o 4 4
2 2
3 3 3 3 3 3
NOTE. Bacteria were incubated with antibiotic; subsequently antibiotic was removed by washing and bacteria were cultured in 7H9 broth, as described in Materials and Methods. * After exposure to antibiotics.
PAE on M. avium growth, 107 bacteria were treated with amikacin at MIC and at 4X the MIC for 30, 60, and 120 min (table 2). The results show that increase of bacterial inoculum from 104 to 107 organisms had no significant impact on the persistence of amikacin effect after exposure of M. avium to MIC or 4X the MIC for 30, 60, or 120 min.
Discussion The study of the PAE provides important information about the action of an antimicrobial agent that cannot be derived from the standard in vitro susceptibility tests, based on the determination of MIC [I]. Previous studies using gram-positive cocci and gram-negative bacilli showed the importance of the PAE in the in vivo activity of an antimicrobial [1, 2]. For example, accumulating evidence suggests that antimicrobials with more prolonged inhibitory effect on the growth ofgram-negative rods appear to be more effective in the therapy of infections in immunosuppressed hosts [3]. The PAE of aminoglycosides and quinolones against microorganisms causing infections in immunosuppressed individuals is likely responsible for the efficacy of these compounds [8, 9]. Clinical observation for a number of years have suggested
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Table 2. Postantibiotic effect (PAE) of amikacin on M. avium strain 10 1 ( 107 organisms) with various concentrations and durations of exposure. Duration of exposure (min). antibiotic (concentration) 30. Control (no antibiotic) Amikacin (MIC) Amikacin (4X MIC) 60. Amikacin (MIC) Amikacin (4X MIC) 120. Amikacin (MIC) Amikacin (4X MIC)
Time (h) to grow Ilog* 40 40 62 50 62 62 68
PAE (h)
o o 22
IO 22
22 28
that once-a-day therapy with active antimicrobials is sufficient to kill or arrest the growth of Mycobacterium tuberculosis in patients with tuberculosis [10]. The efficacy of combined use of two or more active drugs once a day has been attributed to the long rate of multiplication observed with M. tuberculosis. However, the ability of some antibiotics to arrest M. tuberculosis growth may be due to their PAE. Our results show that exposure of M. avium strain 101 to concentrations of amikacin ~8X the MIC is associated with prolonged PAE~ M. avium required up to 78 h to grow I log after being exposed to amikacin for 2 h. In contrast, exposure to rifapentine did not induce significant lag phase of M. avium growth after antibiotic was removed from the medium. These experiments show that PAE is dose- and timedependent and is influenced by the number of bacteria in the inoculum. Although amikacin does not concentrate intracellularly, studies in animal models (evaluating viable intracellular bacteria in liver and spleen), studies in vitro using the macrophage system of intracellular infection, and clinical trials in humans have confirmed its efficacy against M. avium. In the experimental models and clinical trials in humans, the use of amikacin once a day has been shown to be as effective as when given three times a day, suggesting a PAE of the antibiotic on M. avium [11-13]. In addition, recent experimental studies using liposome-encapsulated amikacin have shown release of large intracellular concentrations of the antibiotic [14]. Liposome preparations are currently in clinical trials in humans, making our data on PAE clinically relevant. The precise mechanisms by which different antimicrobials induce PAE are unknown. Because rnacrolides, lincomycins, tetracyclines, chloramphenicol, and rifamycins bind reversibly to ribosomal subunits, PAE with these antibiotics could represent the time required for the drug to diffuse from the
ribosomes. In contrast, the aminoglycoside antibiotics bind irreversibly to ribosomal subunits, and PAE may represent some form ofnonlethal damage. In addition, elegant studies by Lorian et al. [15) using gram-positive cocci and gram-negative bacilli have demonstrated that even subinhibitory concentrations of antibiotics can have profound effect on bacterial morphology and on the ability of bacteria to multiply. However, the influence of the slow rate of growth of organisms such as mycobacteria on the PAE of an antibiotic is unknown. It is possible that, depending on the antimicrobial used, the long generation time of mycobacteria will have a positive or negative effect on the PAE. The clinical importance of the PAE is associated with the dosing regimens. Antimicrobials with more prolonged inhibitory effects against M. avium would require once-a-day administration, probably independent of the serum half-life. It is important, though, to keep in mind that other pharmacologic characteristics of antimycobacterial agents (e.g., tissue concentration) may add to the phenomenon of PAE. Current studies in our laboratory are aimed at investigating the PAE ofmacrolides (e.g., azithromycin and clarithrornycin) that are active against M. avium in vitro and in vivo. Additional information on the PAE of several antimicrobials is likely to improve the design of regimens for the therapy of disseminated M. avium infection in patients with AIDS. References I. Sande MA. Korzeniowski OM, Alegro GM. Brennan RO, Zak 0, Schgeld WM. Intermittent or continuous therapy of experimental meningitis due to Streptococcus pneumoniae in rabbits: preliminary observation on the post-antibiotic effect in vivo. Rev Infect Dis 1981;3:98-109. 2. McDonald PJ, Craig WA, Kunin CM. Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. J Infect Dis 1977; 135:217-33. 3. Gerber AU, Craig WA, Brugger HP, Feller C, Vastola AP, Brandel J. Impact of dosing intervals on activity of gentamicin and ticarcillin against Pseudomonas aeruginosa in granulocytopenic mice. J Infect Dis 1983;147:910-7. 4. Inderlied CB, Kolonoski PT, Wu M. Young LS. Arnikacin, ciprofloxacin and imipenen treatment for disseminated Mycobacterium avium complex infection of beige mice. Antimicrob Agents Chemother 1989;33: 176-80. 5. Inderlied CB, Young LS, Yamada JK. Determination ofin vitro susceptibility of Mycobacterium avium complex isolates to anti-mycobacterial agents by various methods. Antimicrob Agents Chemother 1987;31: 1697-702. 6. Bundtzen RW, Gerber AU. Cohn DL, Craig WA. post-antibiotic suppression of bacterial growth. Rev Infect Dis 1981;3:28-37. 7. Bermudez LE, Young LS. Activities of amikacin, roxithromycin and azithromycin alone or in combination with tumor necrosis factor against Mvcobacterium avium complex. Antimicrob Agents Chemother 1988;32: 1149-53. 8. Mattie H, Craig WA, Pechere JP. Review: determinants of efficacy and toxicity of aminoglycosides. J Antimicrob Chemother 1989;24:28193. 9. Zhanel GG, Davidson RJ. Hoban DJ. Reproducibility of the in-vitro
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NOTE. Bacteria were incubated with antibiotics for 30. 60. or 120 min; antibiotic was subsequently removed by washing. Two experiments were done at each concentration. * After exposure to antibiotics.
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post-antibiotic effect offluoroquinolones against Staphylococcus aureus. J Antimicrob Chemother 1990;26:724-6. 10. Hinshaw He. Treatment of tuberculosis. In: Youmans GP, ed. Tuberculosis. Philadelphia: WB Saunders, 1979:457-88. II. Kesavalu L, Gangadharam PRJ, Purmal VK, Podapati NR, Iseman MD. Chemotherapeutic activity of amikacin against Mycobacterium avium intracellulare [abstract]. In: Program and abstracts: 87th annual meeting ofAmerican Society for Microbiology (Atlanta). Washington, DC: American Society for Microbiology, 1987. 12. Kolonoski PT, Wu M, Petrofsky M, Martinelli J, Young LS, Inderlied CB. Combinations of'arnikacin, azithromycin and clofazimine for the treatment of disseminated Mycobacterium avium complex [abstract 1323]. In: Program and abstracts: 29th Interscience Conference on
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Antimicrobial Agents and Chemotherapy (Houston). Washington, DC: American Society for Microbiology, 1989. 13. Chiu J, Nussbaum J, Bozzette S, et al. Treatment ofdisseminated Mycobacterium avium complex infection in AIDS with amikacin, ethambutol, rifampin, and ciprofloxacin. Ann Intern Med 1990;113:35861. 14. Bermudez LE, Yau-Young AO, Lin JP, CoggerJ, Young LS. Treatment of disseminated Mycobacterium avium complex infection of beige mice with liposome-encapsulated aminoglycosides. J Infect Dis 1990; 161: 1262-8. 15. Lorian V, Ernst J, Amaral L. The post-antibiotic effect defined by bacterial morphology. J Antimicrob Chemother 1989;23:485-91.
Ronald A. Stiller, Irvin L. Paradis, and James H. Dauber
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Pittsburgh School ofMedicine, and Oakland VA Medical Center, Pittsburgh. Pennsylvania
Pneumocystis carinii was recovered from the lungs of a 20-year-old woman in apparent good health who had volunteered to undergo bronchoalveolar lavage (BAL) as a normal control subject. Total and differential cell counts in the BAL fluid revealed a significantly increased number and proportion ofT lymphocytes, although the CD4:CD8 ratio was in the normal range. Despite the lack of specificantibiotic therapy, in a subsequent lavage no P. carinii were recovered, and the total and differential cell counts returned to normal, suggesting that the infection had resolved. Serologic evaluation revealed no evidence of human immunodeficiency virus infection, although elevated titers of antibodies to Epstein-Barr virus were demonstrated, suggesting ongoing or resolving viral infection. These findings suggest that P. carinii may cause subclinical pneumonitis even in the absence of a clinically evident immune deficient state. Furthermore, an increase in cell count and in the proportion of lymphocytes in an otherwise unremarkable BAL may indicate the presence of P. carinii in the airways and may be the only sign of subclinical infection of the respiratory tract by this organism. Pneumocystis carinii has been repeatedly implicated as a pulmonary pathogen in severely ill, malnourished, immunocompromised patients and, most dramatically, in patients with AIDS [I, 2]. On the other hand, scattered communications suggest that P. carinii may also be a pathogen in nonimmunocompromised individuals: Both autopsy series and serologic studies in healthy children have provided considerable
circumstantial evidence that P. carinii can infect normal individuals in the absence of discernible respiratory illness [3, 4]. More recently, P. carinii pneumonia has been reported in 5 elderly patients without predisposing illness [5]. However, we know of no reports documenting the recovery of P. carinii from the lungs of clinically healthy adults.
Case Report Received 13 December 1991; revised 6 April 1992. Written informed consent was obtained and the protocol was approved by the Investigational Review Board of the University of Pittsburgh. Reprints or correspondence: Dr. Ronald A. Stiller, University of Pittsburgh School of Medicine, Division of Pulmonary and Critical Care Medicine, 440 Scaife Hall, Pittsburgh, PA 15261. The Journal of Infectious Diseases 1992;166:926-30 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0039$01.00
A 20-year-old female college student underwent bronchoalveolar lavage (BAL) as a normal volunteer as part of an ongoing study designed to compare the cell profile in BAL fluid from pulmonary transplant recipients with that of clinically healthy adults. She had never smoked, was taking no medication, was sexually inactive, and denied illicit drug use at the time of lavage. Her medical history was remarkable for an uncomplicated course of heterophile antibody-positive infectious mononucleo-
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Subclinical Pneumonitis Due to Pneumocystis carinii in a Young Adult with Elevated Antibody Titers to Epstein-Barr Virus
Postantibiotic Effect of Amikacin and Rifapentine against Mycobacterium avium Complex Luiz E. Bermudez, Martin Wu, Lowell S. Young, and Clark B. Inderlied
Kuzell Institute for Arthritis and Infectious Diseases. Medical Research Institute ofSan Francisco at California Pacific Medical Center; Department ofPathology and Laboratory Medicine. Children's Hospital of Los Angeles. University ofSouthern California
Early clinical experience with penicillin suggested that many infections could be treated adequately with intermittent dosing, even though drug levels in blood and tissue fell below inhibitory concentrations for several hours. Daily administration of only one or two large doses produces high peak and low (sometimes undetectable) trough concentrations of the drug. Studies within the last decade have provided evidence that in many situations pulse dosing works as well as or better than continuous administration of antibiotic, suggesting a postantibiotic effect (PAE) of the drug. The success of intermittent therapy with penicillin was attributed to the persistent effect of the antibiotic on surviving organisms. This persistent suppression of bacterial growth was then attributed to a lag in recovery of growth, a PAE that could take hours. Studies with a number ofgram-positive and gram-negative organisms have demonstrated the PAE of several antibiotics [I, 2). More recently, the efficacy of several antibiotics for treating bacterial infections in immunocompromised patients has been correlated with PAEs [3]. To date, PAE has been measured only against organisms that cause extracellular infections, but it is possible that the ability ofan antibiotic to inhibit growth ofintracellular bacteria is also related to the PAE. For instance, amikacin is active in vitro and in vivo against Mycobacterium avium even though serum levels exceed the MIC for only a few hours [4].
Received 29 January 1992; revised 4 May ·1992. Grant support: National Institutes of Health (AI-72637). Reprints or correspondence: Dr. Luiz E. Bermudez. Kuzell Institute. 2200 Webster s.. Rm. 305. San Francisco. CA 94115. The Journal of Infectious Diseases 1992;166:923-6 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0038$01.00
In this study we compared the PAE ofamikacin and rifapentine (known to concentrate intracellularly) on growth of M. avium.
Materials and Methods Bacteria. M. avium complex strain 101 (serotype 1) was isolated from a patient with AIDS. Strain 101 is virulent, as demonstrated by LD so studies in animals. Bacteria were cultured in Middlebrook agar 7H 10 medium (Difco, Detroit) for 10 days at 37°C. Morphologically similar colonies were harvested and resuspended in 7H9 broth supplemented with OADC (oleic acid, albumin, dextrose, and catalase; Difco). Bacteria were allowed to grow for 5 days. Tubes were maintained in stationary conditions but were slightly agitated by inversion twice a day. The day ofthe experiment, log-phase organisms were washed by centrifugation and resuspended in 7H9 broth containing antibiotics (I X, 4X, 8X MIC). Antibiotics. Amikacin (provided by Bristol Laboratories, Syracuse. NY) was diluted in phosphate buffer, pH 7.2, and stored in portions at a concentration of 1000 Jlg/mL at - 70°C. Rifapentine was provided by Le Petit, Milan, Italy. After dilution, 500 Jlg/mL suspensions were stored at -70°C up to I week. MICs, determined according to previously described protocol [5], were 4 Jlg/mL for amikacin and 2 Jlg/mL for rifapentine. Exposure to antibiotics and removal. At time zero an M. avium suspension containing either 104 or 107 cfu/rnl. was exposed to the antibiotic (10 mL of the bacterial suspension was treated with the drug to obtain the desirable final concentration of the antibiotic). After incubation for 15,30,60, and 120 min at 37°C, the tubes were centrifuged (2000 g. 15 min) and the bacterial pellet was resuspended in PBS. Bacteria were washed three times to ensure complete removal of the antibiotics and, after the third wash, resuspended in 7H9 broth with OADC. Removal of90% of supernatant in two washings reduces antimicrobial concentrations 100-fold [6].
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Postantibiotic effect (PAE) has received little attention in the therapy of chronic intracellular infections, such as those caused by mycobacteria. Amikacin is active therapeutically against Mycobacterium avium complex, even though serum levels exceed the MIC for only a few hours. To determine the PAE of amikacin and rifapentine for M. avium, bacteria were exposed to concentrations of IX, 4X, and lOX the MIC of each drug for up to 120 min. Regrowth of M. avium was compared with similarly diluted untreated cultures. No PAE was observed on an inoculum of 104 bacteria when rifapentine was used at 5X MIC, although a slight inhibition of growth was obtained at tux MIC for 2 h. For amikacin, PAE was observed up to 48 h at concentrations of 4X and sx MIC and exposure times of 30-120 min. A PAE of 22 h was seen with 107 cfu of M. avium during incubation for 30 min with amikacin at 4x MIC. These results show that amikacin, unlike rifapentine, has a long PAE against M. avium.
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Counts of viable bacteria. Counts of viable bacteria were made after plating onto 7H 10 agar plates as previously reported [7]. To obtain uniform bacterial suspensions, resuspended bacterial suspensions were vortex agitated for 1 min before plating. The number of viable organisms was determined at different time points between zero and 96 h after removal of the antibiotic. In some experiments, quantitative plate counts were done before and after centrifugation to rule out an effect of three washes on bacterial counts. No significant difference was observed between the number of viable bacteria before and after centrifugation (data not shown). Definition. PAE was defined as the difference in time required for a culture to increase by I log after antibiotic exposure minus that time for unexposed bacteria.
Effect of exposure to MIC of amikacin and rifapentine on growth of M. avium. To determine if exposure of M. avium strain 101 to MIC of amikacin or rifapentine had any persistent effect on bacterial growth, a suspension of 104 cfu of M. avium was incubated with MICs of amikacin or rifapentine (4 and 2 ~g/mL, respectively) for 30, 60, and 120 min. Antibiotics were then removed by washing, and the bacteria were cultured on medium without antibiotic. As shown in table I, exposure to amikacin for 30 or 60 min was associated with PAE of6 h (36 h to reach I log growth compared with 30 h for bacteria not treated with drugs). Exposure for 120 min was associated with PAE of 10 h. In contrast, exposure of M. avium to rifapentine for 30, 60, or 120 min was not associated with persistent inhibition of growth. Effect ofantibiotic concentration on PAE. Table I shows that the increase in concentration of antibiotics prolonged the persistent effect ofamikacin and rifapentine on M. avium growth. Incubation of M. avium with amikacin for 30 min at a concentration 4X the MIC had a PAE of 18 h, while incubating M. avium with rifapentine (5X the MIC) for 30 min was associated with a PAE of only 4 h. Of note, when the amikacin concentration was increased to 4X or 8X the MIC, there was no significant increase in the PAE. For example, the PAE for bacteria exposed to 4X the MIC ofamikacin for 30 min was 18 h compared with 20 h when the bacteria were exposed to a concentration of 8X the MIC ofamikacin. Similar results were observed when M. avium was exposed to 4X or 8X the MIC ofamikacin for 60 min. A significant increase in PAE was observed only when M. avium was exposed to sx the MIC for 120 min (PAE, 48 h) in comparison to 4X the MIC (PAE, 28 h). Similarly, exposure of M. avium to increased concentrations of rifapentine (lOX MIC) was not associated with increased persistence of antibiotic effect when compared with exposure to rifapentine at Sx the MIC. Effect ofnumbers ofbacteria in inoculum on PA£. To determine whether increased numbers of bacteria in the inoculum would have any effect on the persistence of the amikacin
Table 1. Postantibioticeffect (PAE) ofamikacin and rifapentine on M. avium strain 101 (104 organisms) with various concentra-
tions and durations of exposure. Duration of exposure (min). antibiotic (concentration) 30. Control M. avium (no antibiotic) Amikacin (MIC) Amikacin (4X MIC) Amikacin (8X MIC) Rifapentine (MIC) Rifapentine (5X MIC) Rifapentine ( lOX MIC) 60. Amikacin (MIC) Amikacin (4X MIC) Amikacin (8X MIC) Rifapentine (MIC) Rifapentine (5X MIC) Rifapentine ( IOx MIC) 120. Amikacin (MIC) Amikacin (4X MIC) Amikacin (8X MIC) Rifapentine (MIC) Rifapentine (5X MIC) Rifapentine ( IOx MIC)
Time (h) to grow Ilog* 30 36 48 50 30 30 30 36
PAE (h)
o 6 18 20
No. of experiments
5 3 3 3
o o o
2
6
3
53
23
60
30
30 34 34 40 58 78 30 34 34
o
3 3 2 2 2
4 4
10 28 48
o 4 4
2 2
3 3 3 3 3 3
NOTE. Bacteria were incubated with antibiotic; subsequently antibiotic was removed by washing and bacteria were cultured in 7H9 broth, as described in Materials and Methods. * After exposure to antibiotics.
PAE on M. avium growth, 107 bacteria were treated with amikacin at MIC and at 4X the MIC for 30, 60, and 120 min (table 2). The results show that increase of bacterial inoculum from 104 to 107 organisms had no significant impact on the persistence of amikacin effect after exposure of M. avium to MIC or 4X the MIC for 30, 60, or 120 min.
Discussion The study of the PAE provides important information about the action of an antimicrobial agent that cannot be derived from the standard in vitro susceptibility tests, based on the determination of MIC [I]. Previous studies using gram-positive cocci and gram-negative bacilli showed the importance of the PAE in the in vivo activity of an antimicrobial [1, 2]. For example, accumulating evidence suggests that antimicrobials with more prolonged inhibitory effect on the growth ofgram-negative rods appear to be more effective in the therapy of infections in immunosuppressed hosts [3]. The PAE of aminoglycosides and quinolones against microorganisms causing infections in immunosuppressed individuals is likely responsible for the efficacy of these compounds [8, 9]. Clinical observation for a number of years have suggested
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Results
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lID 1992; 166 (October)
Table 2. Postantibiotic effect (PAE) of amikacin on M. avium strain 10 1 ( 107 organisms) with various concentrations and durations of exposure. Duration of exposure (min). antibiotic (concentration) 30. Control (no antibiotic) Amikacin (MIC) Amikacin (4X MIC) 60. Amikacin (MIC) Amikacin (4X MIC) 120. Amikacin (MIC) Amikacin (4X MIC)
Time (h) to grow Ilog* 40 40 62 50 62 62 68
PAE (h)
o o 22
IO 22
22 28
that once-a-day therapy with active antimicrobials is sufficient to kill or arrest the growth of Mycobacterium tuberculosis in patients with tuberculosis [10]. The efficacy of combined use of two or more active drugs once a day has been attributed to the long rate of multiplication observed with M. tuberculosis. However, the ability of some antibiotics to arrest M. tuberculosis growth may be due to their PAE. Our results show that exposure of M. avium strain 101 to concentrations of amikacin ~8X the MIC is associated with prolonged PAE~ M. avium required up to 78 h to grow I log after being exposed to amikacin for 2 h. In contrast, exposure to rifapentine did not induce significant lag phase of M. avium growth after antibiotic was removed from the medium. These experiments show that PAE is dose- and timedependent and is influenced by the number of bacteria in the inoculum. Although amikacin does not concentrate intracellularly, studies in animal models (evaluating viable intracellular bacteria in liver and spleen), studies in vitro using the macrophage system of intracellular infection, and clinical trials in humans have confirmed its efficacy against M. avium. In the experimental models and clinical trials in humans, the use of amikacin once a day has been shown to be as effective as when given three times a day, suggesting a PAE of the antibiotic on M. avium [11-13]. In addition, recent experimental studies using liposome-encapsulated amikacin have shown release of large intracellular concentrations of the antibiotic [14]. Liposome preparations are currently in clinical trials in humans, making our data on PAE clinically relevant. The precise mechanisms by which different antimicrobials induce PAE are unknown. Because rnacrolides, lincomycins, tetracyclines, chloramphenicol, and rifamycins bind reversibly to ribosomal subunits, PAE with these antibiotics could represent the time required for the drug to diffuse from the
ribosomes. In contrast, the aminoglycoside antibiotics bind irreversibly to ribosomal subunits, and PAE may represent some form ofnonlethal damage. In addition, elegant studies by Lorian et al. [15) using gram-positive cocci and gram-negative bacilli have demonstrated that even subinhibitory concentrations of antibiotics can have profound effect on bacterial morphology and on the ability of bacteria to multiply. However, the influence of the slow rate of growth of organisms such as mycobacteria on the PAE of an antibiotic is unknown. It is possible that, depending on the antimicrobial used, the long generation time of mycobacteria will have a positive or negative effect on the PAE. The clinical importance of the PAE is associated with the dosing regimens. Antimicrobials with more prolonged inhibitory effects against M. avium would require once-a-day administration, probably independent of the serum half-life. It is important, though, to keep in mind that other pharmacologic characteristics of antimycobacterial agents (e.g., tissue concentration) may add to the phenomenon of PAE. Current studies in our laboratory are aimed at investigating the PAE ofmacrolides (e.g., azithromycin and clarithrornycin) that are active against M. avium in vitro and in vivo. Additional information on the PAE of several antimicrobials is likely to improve the design of regimens for the therapy of disseminated M. avium infection in patients with AIDS. References I. Sande MA. Korzeniowski OM, Alegro GM. Brennan RO, Zak 0, Schgeld WM. Intermittent or continuous therapy of experimental meningitis due to Streptococcus pneumoniae in rabbits: preliminary observation on the post-antibiotic effect in vivo. Rev Infect Dis 1981;3:98-109. 2. McDonald PJ, Craig WA, Kunin CM. Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. J Infect Dis 1977; 135:217-33. 3. Gerber AU, Craig WA, Brugger HP, Feller C, Vastola AP, Brandel J. Impact of dosing intervals on activity of gentamicin and ticarcillin against Pseudomonas aeruginosa in granulocytopenic mice. J Infect Dis 1983;147:910-7. 4. Inderlied CB, Kolonoski PT, Wu M. Young LS. Arnikacin, ciprofloxacin and imipenen treatment for disseminated Mycobacterium avium complex infection of beige mice. Antimicrob Agents Chemother 1989;33: 176-80. 5. Inderlied CB, Young LS, Yamada JK. Determination ofin vitro susceptibility of Mycobacterium avium complex isolates to anti-mycobacterial agents by various methods. Antimicrob Agents Chemother 1987;31: 1697-702. 6. Bundtzen RW, Gerber AU. Cohn DL, Craig WA. post-antibiotic suppression of bacterial growth. Rev Infect Dis 1981;3:28-37. 7. Bermudez LE, Young LS. Activities of amikacin, roxithromycin and azithromycin alone or in combination with tumor necrosis factor against Mvcobacterium avium complex. Antimicrob Agents Chemother 1988;32: 1149-53. 8. Mattie H, Craig WA, Pechere JP. Review: determinants of efficacy and toxicity of aminoglycosides. J Antimicrob Chemother 1989;24:28193. 9. Zhanel GG, Davidson RJ. Hoban DJ. Reproducibility of the in-vitro
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NOTE. Bacteria were incubated with antibiotics for 30. 60. or 120 min; antibiotic was subsequently removed by washing. Two experiments were done at each concentration. * After exposure to antibiotics.
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post-antibiotic effect offluoroquinolones against Staphylococcus aureus. J Antimicrob Chemother 1990;26:724-6. 10. Hinshaw He. Treatment of tuberculosis. In: Youmans GP, ed. Tuberculosis. Philadelphia: WB Saunders, 1979:457-88. II. Kesavalu L, Gangadharam PRJ, Purmal VK, Podapati NR, Iseman MD. Chemotherapeutic activity of amikacin against Mycobacterium avium intracellulare [abstract]. In: Program and abstracts: 87th annual meeting ofAmerican Society for Microbiology (Atlanta). Washington, DC: American Society for Microbiology, 1987. 12. Kolonoski PT, Wu M, Petrofsky M, Martinelli J, Young LS, Inderlied CB. Combinations of'arnikacin, azithromycin and clofazimine for the treatment of disseminated Mycobacterium avium complex [abstract 1323]. In: Program and abstracts: 29th Interscience Conference on
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Antimicrobial Agents and Chemotherapy (Houston). Washington, DC: American Society for Microbiology, 1989. 13. Chiu J, Nussbaum J, Bozzette S, et al. Treatment ofdisseminated Mycobacterium avium complex infection in AIDS with amikacin, ethambutol, rifampin, and ciprofloxacin. Ann Intern Med 1990;113:35861. 14. Bermudez LE, Yau-Young AO, Lin JP, CoggerJ, Young LS. Treatment of disseminated Mycobacterium avium complex infection of beige mice with liposome-encapsulated aminoglycosides. J Infect Dis 1990; 161: 1262-8. 15. Lorian V, Ernst J, Amaral L. The post-antibiotic effect defined by bacterial morphology. J Antimicrob Chemother 1989;23:485-91.
Ronald A. Stiller, Irvin L. Paradis, and James H. Dauber
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Pittsburgh School ofMedicine, and Oakland VA Medical Center, Pittsburgh. Pennsylvania
Pneumocystis carinii was recovered from the lungs of a 20-year-old woman in apparent good health who had volunteered to undergo bronchoalveolar lavage (BAL) as a normal control subject. Total and differential cell counts in the BAL fluid revealed a significantly increased number and proportion ofT lymphocytes, although the CD4:CD8 ratio was in the normal range. Despite the lack of specificantibiotic therapy, in a subsequent lavage no P. carinii were recovered, and the total and differential cell counts returned to normal, suggesting that the infection had resolved. Serologic evaluation revealed no evidence of human immunodeficiency virus infection, although elevated titers of antibodies to Epstein-Barr virus were demonstrated, suggesting ongoing or resolving viral infection. These findings suggest that P. carinii may cause subclinical pneumonitis even in the absence of a clinically evident immune deficient state. Furthermore, an increase in cell count and in the proportion of lymphocytes in an otherwise unremarkable BAL may indicate the presence of P. carinii in the airways and may be the only sign of subclinical infection of the respiratory tract by this organism. Pneumocystis carinii has been repeatedly implicated as a pulmonary pathogen in severely ill, malnourished, immunocompromised patients and, most dramatically, in patients with AIDS [I, 2]. On the other hand, scattered communications suggest that P. carinii may also be a pathogen in nonimmunocompromised individuals: Both autopsy series and serologic studies in healthy children have provided considerable
circumstantial evidence that P. carinii can infect normal individuals in the absence of discernible respiratory illness [3, 4]. More recently, P. carinii pneumonia has been reported in 5 elderly patients without predisposing illness [5]. However, we know of no reports documenting the recovery of P. carinii from the lungs of clinically healthy adults.
Case Report Received 13 December 1991; revised 6 April 1992. Written informed consent was obtained and the protocol was approved by the Investigational Review Board of the University of Pittsburgh. Reprints or correspondence: Dr. Ronald A. Stiller, University of Pittsburgh School of Medicine, Division of Pulmonary and Critical Care Medicine, 440 Scaife Hall, Pittsburgh, PA 15261. The Journal of Infectious Diseases 1992;166:926-30 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6604-0039$01.00
A 20-year-old female college student underwent bronchoalveolar lavage (BAL) as a normal volunteer as part of an ongoing study designed to compare the cell profile in BAL fluid from pulmonary transplant recipients with that of clinically healthy adults. She had never smoked, was taking no medication, was sexually inactive, and denied illicit drug use at the time of lavage. Her medical history was remarkable for an uncomplicated course of heterophile antibody-positive infectious mononucleo-
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Subclinical Pneumonitis Due to Pneumocystis carinii in a Young Adult with Elevated Antibody Titers to Epstein-Barr Virus
